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Subjects

Abstract

Enteric nervous system neuropathy causes a wide range of severe gut motility disorders. Cell replacement of lost neurons using enteric neural stem cells (ENSC) is a possible therapy for these life-limiting disorders. Here we show rescue of gut motility after ENSC transplantation in a mouse model of human enteric neuropathy, the neuronal nitric oxide synthase (nNOS−/−) deficient mouse model, which displays slow transit in the colon. We further show that transplantation of ENSC into the colon rescues impaired colonic motility with formation of extensive networks of transplanted cells, including the development of nNOS+ neurons and subsequent restoration of nitrergic responses. Moreover, post-transplantation non-cell-autonomous mechanisms restore the numbers of interstitial cells of Cajal that are reduced in the nNOS−/− colon. These results provide the first direct evidence that ENSC transplantation can modulate the enteric neuromuscular syncytium to restore function, at the organ level, in a dysmotile gastrointestinal disease model.

Introduction

Neuropathological loss within the enteric nervous system (ENS) has been implicated in a wide range of severe gut motility disorders, such as achalasia1,2,3, gastroparesis4,5, slow transit constipation6,7,8 and Hirschsprung’s disease (aganglionic megacolon)9,10, as well as being associated with a number of central nervous system disorders11,12,13. Potential replacement of lost neurons using stem cell replacement is an attractive therapy for such life-limiting disorders. Enteric neural stem cells (ENSC), which exist in both embryonic and adult gut, have been suggested as potential cell source for such treatments14,15. We and others have previously demonstrated the potential of both mouse16,17 and human18 ENSC to integrate within wild-type ganglionated mouse colon. Yet a limiting factor in the advancement of ENSC therapies for human application has been the failure to demonstrate functional rescue of motility in pathological disease models. Recent studies have demonstrated the successful integration of murine and human ENSC within aganglionic colon both in vivo16,19 and ex vivo20,21; however, the severity of the gut phenotype and poor survival of homozygote mice has limited their in vivo use for investigating the potential functional rescue, at the organ level, of ENSC-based therapies.

Other models of neuronal loss are, therefore, essential to test the viability of cell-based transplantation techniques to restore functional deficits resulting from neuropathology. The loss of neuronal nitric oxide synthase (nNOS) has been implicated in a range of human enteric neuropathies22, including oesophageal achalasia23, infantile hypertrophic pyloric stenosis24, gastroparesis (idiopathic and diabetic)25, colonic dysfunction26 and Hirschsprung’s disease27,28. Notably, nNOS−/− mice recapitulate the clinical phenotype of a number of human diseases displaying both delayed gastric emptying29,30,31, and slow transit in the colon32 hence providing an ideal model to establish if ENSC can restore function after in vivo transplantation. Here we show rescue of motility, after transplantation of ENSC, within the nNOS−/− mouse colon. We further demonstrate robust restoration of nitrergic responses coincident with the development of nNOS+ neurons in an nNOS-deficient microenvironment. In addition, we show concurrent rescue of interstitial cells of Cajal (ICC) within the nNOS−/− colon after ENSC transplantation. Thus, we propose that ENSC can modulate the neuromuscular syncytium via both cell-autonomous and non-cell-autonomous mechanisms to restore function, at the organ level, and ultimately rescue motility.

Results

Transplanted ENSC extensively integrate in nNOS−/− colon

To isolate ENSC, we used donor Wnt1cre/+;R26RYFP/YFP mice (P2–P7), in which neural crest cells and their enteric derivatives express endogenous yellow fluorescent protein (YFP). This endogenous expression allowed for isolation and fate-mapping of labelled donor ENSC. Selected YFP+ cells maintained expression and formed characteristic neurospheres within 1 month in culture (Supplementary Fig. 1). To assess the composition of neurospheres, immunohistochemistry and qRT–PCR were performed to establish the presence of typical ENS cell types. Such neurospheres were found to express ENS markers such as the pan-neuronal marker TuJ1 (Supplementary Fig. 1a), the neural crest progenitor marker SOX10 (Supplementary Fig. 1b) and the glial marker S100 (Supplementary Fig. 1c). Notably, in addition to multipotent neural crest progenitors, neuronal markers, including NOS+ neurons (Supplementary Fig. 1e–g), were observed within neurospheres in vitro. Hence, we sought to establish the ability of such neurospheres to colonize and populate nNOS−/− colon in vivo. As opposed to wild-type colon, which contains nNOS+ cell bodies and fibres (Fig. 1a), nNOS−/− mice display complete loss of nNOS+ neurons in the colon (Fig. 1b).

We transplanted three YFP+ neurospheres (∼6 × 104 cells in total) into the distal colon of nNOS−/− mice at P14–P17 via laparotomy. Live imaging analysis, 4 weeks after transplantation, revealed the presence of extensive anastomosing networks of transplanted YFP+ cells colonizing, on average, 5.46±0.5 mm2 (n=10) of distal colon at the site of transplantation (Fig. 1c). Subsequent immunohistochemistry revealed more extensive networks of GFP+ cells (Supplementary Figs 2 and 3). GFP+ filamentous networks could be observed extending in both oral and aboral directions from the site of transplantation (Supplementary Fig. 2 and Supplementary Movie 1) including integration within the proximal colon. Post-acquisition mapping of transplanted cells revealed the largest continuous GFP+ network extending 10.79 mm (Supplementary Fig. 2b). Along the length of the colon GFP+ cells were found to co-express the neuronal marker TuJ1 (Fig. 1d–f) and project fibres (Fig. 1d, arrowheads), which contacted the endogenous neuronal network at the level of the myenteric plexus. GFP+ cells were also identified within endogenous myenteric ganglia (Fig. 1g–i), where fine GFP+ fibres and varicosities were observed encompassing and tracing the path of endogenous neuronal fibre tracts (Fig. 1g, arrowheads). Confocal imaging of the entire colon also revealed GFP+ cells co-expressing TuJ1 integrated within ganglia along the length of the colon (Supplementary Fig. 3 and Supplementary Movie 2) up to a maximum of 42.4 mm from the site of transplantation thus confirming the ability of transplanted cells to migrate within the tunica muscularis. Transplanted cells displayed enteric neuronal characteristics including integration of bipolar (Supplementary Fig. 4a–d) and multipolar GFP+ cells (Supplementary Fig. 4e,f) phenocopying the morphology of enteric interneurons and motor neurons, respectively.

Transplanted ENSC regenerate nNOS+ neurons

To determine if transplanted ENSC have the capacity to develop an NOS+ phenotype in vivo similar to in vitro cultures, immunohistochemistry and RT–PCR were performed. Within the distal colon, transplanted YFP+ cells co-expressed both the neuronal marker TuJ1 and the neuronal nitric oxide synthase marker nNOS (Fig. 2a–d). nNOS+ neurons were identified within ganglia-like structures (Fig. 2c,d, arrowheads) extending multiple nNOS+ projections (Fig. 2c,d, arrows) within the network of transplanted cells. The presence of nNOS+ neurons was further confirmed with PCR analysis demonstrating the specific expression of the nNOS transcript within transplanted colon compared with the complete absence of transcript in non-transplanted tissues (Fig. 2e).

To assess the proliferative capacity of transplanted cells, BrdU was applied 24 h post surgery and incorporation was assessed at 4 weeks. Incorporation of BrdU was observed within transplanted cells co-expressing TuJ1 (Fig. 2f–i, arrows) or nNOS (Fig. 2j–m, arrows) suggesting that transplanted ENSC have the ability to proliferate at early post-transplantation stages and subsequently differentiate to form mature neurons including nNOS+ neurons within an nNOS-deficient microenvironment.

To establish if the restored relaxatory response was due to the presence of transplanted nNOS+ neurons, EFS-induced responses observed in non-adrenergic non-cholinergic conditions were analysed in the presence and absence of the nitric oxide synthase blocker L-NAME. L-NAME significantly reduced this EFS-induced relaxation (−0.74±0.17 g s versus −0.12±0.16 g s; n=4; P=0.0389, Student’s t-test) in transplanted nNOS−/− colon (Fig. 3g–i). Taken together, these results indicate that transplantation of ENSC and development of nNOS+ neurons in the nNOS−/− distal colon results in partial restoration of nitrergic responses.

ENSC transplantation increases basal contractile properties

Organ bath physiology also revealed large amplitude basal contractions in transplanted nNOS−/− distal colonic segments (Fig. 3d). To investigate these responses, basal contractile patterns were recorded in both the distal and proximal colonic segments in control conditions (Krebs solution), and after the addition of individual neurotransmitter antagonists or tetrodotoxin (TTX).

Upper GI transit is not affected by ENSC transplantation

As nNOS−/− mice display pan-enteric deficits in nNOS signalling and have been shown to have delayed gastric emptying, we sought to assess if transplantation to the distal colon could affect intestinal transit parameters outside of the colonic region. Using fluorescent in vivo imaging, liquid stomach emptying at 30 min (Fig. 5c,d) was significantly delayed in nNOS−/− mice (32.9±5.9%, n=3) compared to C57BL/6J mice (56.4±4.6%; n=3; P=0.0316, Student’s t-test) similar to previously described studies. Notably, no difference was observed in liquid stomach emptying between control nNOS−/−, sham-operated and transplanted nNOS−/− group means as determined by one-way ANOVA (F(2,6)=1.00, P=0.421; Fig. 5c,d).

To assess partial intestinal transit, mice were killed 90 min after gavage of a fluorescent dye. Fluorescent imaging was performed ex vivo and the distance the dye had transited was calculated as a percentage of total intestinal length (Fig. 5e,f). nNOS−/− mice displayed reduced intestinal transit (79.9±2.1%, n=3) compared to C57BL/6J mice (89.1±1.1%, n=3; P=0.0176, Student’s t-test). Again, there was no difference between control nNOS−/−, sham-operated and transplanted nNOS−/− transit distance at 90 min as determined by one-way ANOVA (F(2,6)=0.56, P=0.6). We conclude that as both stomach emptying and partial intestinal transit time are unaffected by transplantation, the overall improvement in total intestinal transit time in transplanted nNOS−/− mice is due to substantial increases in colonic transit.

ENSC transplantation restores ICC numbers

We next sought to ensure that the changes in GI function following transplantation were not secondary to other phenomena such as inflammation. On initial dissection, and after careful analysis, no significant changes in the gross anatomy of the colon or evidence of inflammatory responses were observed (Supplementary Fig. 7a). In addition, no inflammation was observed on histological examination (Supplementary Fig. 7b). Histological analysis also revealed that there were no differences in either colonic diameter (P>0.05; Supplementary Fig. 7c,e) or muscle thickness (P>0.05; Supplementary Fig. 7d,f) when comparing both the proximal and distal colon in nNOS−/−, sham-operated nNOS−/− and transplanted nNOS−/− animals by one-way ANOVA.

Discussion

Recent studies have demonstrated the functional integration of both mouse and human ENSC-derived neurons in wild-type mouse colon after in vivo transplantation16,17,18. Functional analysis in these animals that lack a neuropathological phenotype is restricted to individual or groups of transplanted neurons rather than assessment of colonic physiology. We now report that transplantation of a population of selected enteric neural crest-derived cells has a clear and positive functional impact, rescuing motility in a pathophysiological mouse model that recapitulates the phenotype of several clinically relevant human disorders. We demonstrate that this rescue is achieved through both cell-autonomous restoration of nitrergic responses that were absent, and non-cell-autonomous rescue of ICC numbers, which were also found to be deficient in the nNOS−/− colon.

The ability to form nitrergic neurons is a critical step in the development of ‘normal’ enteric circuitry and many enteric disorders would likely benefit from the transplantation and engraftment of nNOS+ cells. In clearly demonstrating the potential to restore nNOS neurons after ENSC transplantation, which to our knowledge is the first study to show functional effects at the organ level, we believe ENSC therapies could impact widely at the clinical level. Interestingly, transplantation of ENSC led to restoration of nitrergic responses and increases in post-stimulation ‘rebound’ contraction. Previous studies have suggested that this post-stimulation contraction is mediated via nitric oxide33 or through a generalized inhibitory response via eicosanoids rather than linked to a distinct inhibitory transmitter34. The finding that transplantation of ENSC cells restored nitrergic inhibitory responses and altered post-stimulus responses suggests adaptation of neuromuscular signalling mechanisms at multiple levels. Of note, there are differences in the methodology of our study and that of these earlier studies, hence further investigation is required to assess the functional interaction of transplanted ENSC at the cellular level. Furthermore, the novel finding that ENSC transplantation rescues ICC numbers in the nNOS−/− colon in a non-cell-autonomous fashion raises interesting questions regarding the interaction of transplanted ENSC with associated cells within intestinal tissues. Intestinal excitation–contraction coupling is extremely complex, with GI smooth muscle receiving inputs from multiple excitable cells. ICC exist within extensive networks throughout the intestinal tract35 exhibiting pacemaker activity36,37,38 and transducing neural signals to the smooth muscle39,40. Recent studies show that ICC are innervated by nitrergic nerves41 and express nitric oxide sensitive guanylate cyclase in both guinea pig42 and mouse colon43 and that disruptions in nNOS signalling can result in loss of ICC in the stomach25,44. The findings of similar disruptions in ICC networks, in the present study, along the length of the nNOS−/− colon further emphasize the link between NOS signalling and ICC development. Our previous work has demonstrated that the selection of ENSC, as in this study, excludes incorporation of mesenchyme-derived ICC45. Notably, previous studies have suggested that enteric neurons are the dominant source of SCF, the natural ligand for c-Kit46, and that 60% of nNOS+ enteric neurons express SCF47 possibly providing a direct signalling link between enteric neurons and ICC. The finding that ENSC-derived neurospheres express SCF ligand in line with these earlier reports provides a possible mechanism whereby ICC modification post transplantation is likely through SCF signalling from transplanted neurons.

One of the challenges for cell replenishment therapies is scaling up for potential human application. It remains possible that significant cell numbers will be required to facilitate functional outcomes in human patients. A recent study has demonstrated derivation of enteric neural crest from human pluripotent stem cells and potential rescue of a Hirschsprung phenotype48. As opposed to our study, this investigation transplanted up to 4 million cells to Ednrbs−l/s−l (SSL/LEJ) colon. While transplantation led to survival of mice, no mechanism was presented of the graft-mediated host rescue. The findings of our study suggest that in addition to engraftment of neural crest cells, non-cell-autonomous modification of the neuromuscular apparatus may be responsible for this rescue. Our study thus highlights the potential to apply a limited number of cells, to a particular area, which subsequently could impact on function throughout the organ and have significant clinical benefits. In addition, we demonstrate the potential for collecting postnatal tissue for use in transplantation studies and the ability of cells from this source to restore function. This caveat has significant therapeutic benefits as ENSC could be collected autologously or from matched donors to limit the potential for immunological rejection. Moreover, previous long-term safety studies using identical postnatal ENSC have demonstrated long-term survival of ENSC-derived cells restricted only to the region of transplantation16, which may provide a substantial benefit over the potential therapeutic application of cells derived from pluripotent sources.

We conclude that this study provides the first evidence that ENSC can rescue GI motility within a neuropathic model and may provide the basis for development of targeted cellular therapies for enteric neuropathies.

Methods

Animals

Male and female Wnt1cre/+;R26RYFP/YFP mice, in which neural crest cells express YFP, were used as donors to obtain YFP+ ENSC. Heterozygote nNOS (B6.129S4-Nos1tm1Plh/J) mice were obtained from The Jackson Laboratory (Bar Harbor, MN, USA). Male and female homozygote nNOS knockout (nNOS−/−) mice were bred and maintained for use as recipients. Four–5-week-old C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, MN, USA) and killed as age-matched controls at 6 weeks. Animals used for these studies were maintained, and the experiments performed, in accordance with the UK Animals (Scientific Procedures) Act 1986 and approved by the University College London Biological Services Ethical Review Process. Animal husbandry at UCL Biological Services was in accordance with the UK Home Office Certificate of Designation.

Cell isolation and enrichment

The entire small intestine and colon was obtained from early postnatal (P2–P7) Wnt1cre/+;R26RYFP/YFP mice after cervical dislocation, and removed to sterile PBS for further dissection. Jejunum, ileum and colon muscle strips were obtained following removal of the mucosa via fine dissection. Intestinal cells were dissociated and YFP+ cells isolated using fluorescence-activated cell sorting with a MoFloXDP cell sorter (Beckman Coulter, UK). YFP positive (YFP+) cells were selected using a 530/40 filter set. Gating parameters were set using cells from wild-type gut and applied to increase specificity of selection of YFP+ cells.

In vivo ENSC transplantation

YFP-expressing neurospheres derived from Wnt1cre/+;R26RYFP/YFP mice were transplanted into the distal colon of P14–P17 nNOS−/−, via laparotomy under isoflurane anaesthetic. Briefly, the distal colon was exposed and a small pocket was created in the tunica muscularis with the bevel of a 30G needle. A neurosphere, containing ∼2 × 104 YFP+ cells, was subsequently transplanted to this site by mouth pipette using a pulled glass micropipette. Each transplanted tissue typically received three neurospheres (∼6 × 104 YFP+ cells in total). Transplanted nNOS−/− mice were typically maintained for 4 weeks post transplantation before killing and removal of the colon for analysis. ‘Sham’ operations were performed as controls in which the intestine was manipulated in an identical fashion without the addition of YFP+ cells.

qRT–PCR

Total RNA was isolated from three pooled neurospheres at the time of surgery using an RNeasy Micro Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. First-strand cDNA was amplified from 100 ng RNA using SuperScript VILO cDNA Synthesis Kit (Life Technologies Ltd, Paisley, UK). RT quantitative PCR was performed with an ABI Prism 7500 sequence detection system (Applied Biosystems) using the Quantitect SYBR Green PCR kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. qRT–PCR was performed in triplicate using region-specific primers for GAPDH, TuJ1, SOX10 and S100 (Supplementary Table 1). Gene expression data were expressed as a proportion of GAPDH housekeeping gene, as a reference, using a 1/ΔCt calculation.

In vivo transit analysis

To test GI transit, either 100 μl Gastrosense 750 (Perkin Elmer, USA) or 100 μl Brilliant Blue FCT (E122) solution (Langdales, UK) was administered to the stomach via gavage at 6 weeks (4 weeks post transplantation). Total GI transit time was calculated from time of administration to the first visualization of dye in the stool. Stool output in 1 h was calculated as: total number of stool in 1 h × stool weight. To assess stomach emptying and partial transit time, Gastrosense 750 fluorescence was imaged using an IVIS Lumina III In Vivo Imaging System (Perkin Elmer, USA). At 30 min, in vivo images were obtained and percentage stomach emptying calculated as:

Statistical analysis

Data are expressed as mean±s.e.m. Differences in the data were evaluated between nNOS−/− control, sham-operated and transplanted groups using one-way ANOVA and subsequent intergroup differences were determined by unpaired Student’s t-test. P values <0.05 were taken as statistically significant. The ‘n values’ reported refer to the number of mice or colonic segments used for each protocol. Each muscle was taken from a separate animal.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files or from the corresponding author on reasonable request.

Acknowledgements

The authors thank Dr Ayad Eddaoudi, Ms Stephanie Canning (UCL Great Ormond Street Institute of Child Health Flow Cytometry Facility) and Dr Dale Moulding (UCL Great Ormond Street Institute of Child Health Imaging Facility) for technical support. The authors also gratefully acknowledge use of the In Vivo Imaging services at the UCL Great Ormond Street Institute of Child Health, funded by The Alternative Hair Charitable Foundation. All research at Great Ormond Street Hospital NHS Foundation Trust and UCL Great Ormond Street Institute of Child Health are made possible by the NIHR Great Ormond Street Hospital Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. This project has received some funding from the European Union's Horizon 2020 research and innovation programme ‘INtestinal Tissue Engineering Solution’ under grant No 668294. N.T. is supported by Great Ormond Street Hospital Children’s Charity (GOSHCC—V1258). C.J.M., J.E.C. and D.N. were funded through a GOSHCC grant (W1018C) awarded to N.T. (Principal Investigator) and A.J.B. (Co-Investigator). J.E.C. was part-funded by a grant from the Medical Research Council (G0800973) awarded to N.T. (Principal Investigator) and A.J.B. (Co-Investigator).

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